Biofilms are antibiotic-resistant bacterial aggregates that grow on moist surfaces and can trigger hospitalacquired infections. They provide a classical example in biology where the dynamics of cellular communities may be observed and studied. Gene expression regulates cell division and differentiation, which affect the biofilm architecture. Mechanical and chemical processes shape the resulting structure. We gain insight into the interplay between cellular and mechanical processes during biofilm development on air-agar interfaces by means of a hybrid model. Cellular behavior is governed by stochastic rules informed by a cascade of concentration fields for nutrients, waste, and autoinducers. Cellular differentiation and death alter the structure and the mechanical properties of the biofilm, which is deformed according to Föppl-Von Kármán equations informed by cellular processes and the interaction with the substratum. Stiffness gradients due to growth and swelling produce wrinkle branching. We are able to reproduce wrinkled structures often formed by biofilms on air-agar interfaces, as well as spatial distributions of differentiated cells commonly observed with B. subtilis.

Environmental bacteria are most often endowed with native surface-attachment programs that frequently conflict with efforts to engineer biofilms and synthetic communities with given tridimensional architectures. In this work, we report the editing of the genome of Pseudomonas putida KT2440 for stripping the cells of most outer-facing structures of the bacterial envelope that mediate motion, binding to surfaces, and biofilm formation. To this end, 23 segments of the P. putida chromosome encoding a suite of such functions were deleted, resulting in the surface-naked strain EM371, the physical properties of which changed dramatically in respect to the wild type counterpart. As a consequence, surface-edited P. putida cells were unable to form biofilms on solid supports and, because of the swimming deficiency and other alterations, showed a much faster sedimentation in liquid media. Surface-naked bacteria were then used as carriers of interacting partners (e.g., Jun–Fos domains) ectopically expressed by means of an autotransporter display system on the now easily accessible cell envelope. Abstraction of individual bacteria as adhesin-coated spherocylinders enabled rigorous quantitative description of the multicell interplay brought about by thereby engineered physical interactions. The model was then applied to parametrize the data extracted from automated analysis of confocal microscopy images of the experimentally assembled bacterial flocks for analyzing their structure and distribution. The resulting data not only corroborated the value of P. putida EM371 over the parental strain as a platform for display artificial adhesins but also provided a strategy for rational engineering of catalytic communities.

The often striking macroscopic patterns developed by motile bacterial populations on agar plates are a consequence of the environmental conditions where the cells grow and spread. Parameters such as medium stiffness and nutrient concentration have been reported to alter cell swimming behavior, while mutual interactions among populations shape collective patterns. One commonly observed occurrence is the mutual inhibition of clonal bacteria when moving toward each other, which results in a distinct halt at a finite distance on the agar matrix before having direct contact. The dynamics behind this phenomenon (i.e., intolerance to mix in time and space with otherwise identical others) has been traditionally explained in terms of cell-to-cell competition/cooperation regarding nutrient availability. In this work, the same scenario has been revisited from an alternative perspective: the effect of the physical mechanics that frame the process, in particular the consequences of collisions between moving bacteria and the semi-solid matrix of the swimming medium. To this end, we set up a simple experimental system in which the swimming patterns of Pseudomonas putida were tested with different geometries and agar concentrations. A computational analysis framework that highlights cell-to-medium interactions was developed to fit experimental observations. Simulated outputs suggested that the medium is compressed in the direction of the bacterial front motion. This phenomenon generates what was termed a compression wave that goes through the medium preceding the swimming population and that determines the visible high-level pattern. Taken together, the data suggested that the mechanical effects of the bacteria moving through the medium created a factual barrier that impedes to merge with neighboring cells swimming from a different site. The resulting divide between otherwise clonal bacteria is thus brought about by physical forces—not genetic or metabolic programs.

Biofilms are multicellular bacterial structures that adhere to surfaces and often endow the bacterial population with tolerance to antibiotics and other environmental insults. Biofilms frequently colonize the tubing of medical devices through mechanisms that are poorly understood. Here we studied the helicoidal spread of Pseudomonas putida biofilms through cylindrical conduits of varied diameters in slow laminar flow regimes. Numerical simulations of such flows reveal vortical motion at stenoses and junctions, which enhances bacterial adhesion and fosters formation of filamentous structures. Formation of long, downstream-flowing bacterial threads that stem from narrowings and connections was detected experimentally, as predicted by our model. Accumulation of bacterial biomass makes the resulting filaments undergo a helical instability. These incipient helices then coarsened until constrained by the tubing walls, and spread along the whole tube length without obstructing the flow. A three-dimensional discrete filament model supports this coarsening mechanism and yields simulations of helix dynamics in accordance with our experimental observations. These findings describe an unanticipated mechanism for bacterial spreading in tubing networks which might be involved in some hospital-acquired infections and bacterial contamination of catheters.

Agglomeration processes occur in many different realms of science such as colloid and aerosol formation or formation of bacterial colonies. We study the influence of primary particle density in agglomerate structure using diffusion-controlled Monte Carlo simulations with realistic space scales through different regimes (DLA and DLCA). The equivalence of Monte Carlo time steps to real time scales is given by Hirsch's hydrodynamical theory of Brownian motion. Agglomerate behavior at different time stages of the simulations suggests that three indices (fractal exponent, coordination number and eccentricity index) characterize agglomerate geometry. Using these indices, we have found that the initial density of primary particles greatly influences the final structure of the agglomerate as observed in recent experimental works.

Adaptive laboratory evolution (ALE) is a general and effective strategy for optimizing the design of engineered genetic circuits and upgrading metabolic phenotypes. However, the specific characteristics of each microorganism typically ask for exclusive conditions that need to be adjusted to the biological chassis at stake. In this work, we have adopted a doit-yourself (DIY) approach to implement a flexible and automated framework for performing ALE experiments with the environmental bacterium and metabolic engineering platform Pseudomonas putida. The setup includes a dual-chamber semi-continuous logphase bioreactor design combined with an anti-biofilm layout to manage specific traits of this bacterium in long-term cultivation experiments. As a way of validation, the prototype was instrumental for selecting fast-growing variants of a P. putida strain engineered to metabolize D-xylose as sole carbon and energy source after running an automated 42 days protocol of iterative regrowth. Several genomic changes were identified in the evolved population that pinpointed the role of RNA polymerase in controlling overall physiological conditions during metabolism of the new carbon source.

Exploitation of biofilms for industrial processes requires them to adopt suitable physical structures for rendering them efficient and predictable. While hydrodynamics could be used to control material features of biofilms of the platform strain Pseudomonas putida KT2440 there is a dearth of experimental data on surface-associated growth behavior in such settings. Millimeter scale biofilm patterns formed by its parental strain P. putida mt-2 under different Reynolds numbers (Re) within laminar regime were analyzed using an upscale experimental continuous cultivation assembly. A tile-scan image acquisition process combined with a customized image analysis revealed patterns of dense heterogeneous structures at Re = 1000, but mostly flattened coverings sparsely patched for Re < 400. These results not only fix the somewhat narrow hydrodynamic regime under which P. putida cells form stable coatings on surfaces destined for large-scale processes, but also provide useful sets of parameters for engineering catalytic biofilms based on this important bacterium as a cell factory.

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